1. Field of the Invention
The present invention relates to a method for transforming the genus Pinus with Agrobacterium tumefaciens. More particularly, the present invention relates to the transformation of Pinus species with strains of A. tumefaciens which harbor those Ti plasmids which are capable of causing crown gall in Pinus or derivatives thereof.
2. Description of the Prior Art
A. tumefaciens is a soil bacterium that causes crown gall on plants by infecting wounds that are generally below or at the soil surface; however, aerial plant parts can also be infected. A large tumor-inducting (Ti) plasmid is essential for tumor formation in the infected plant. A portion of the Ti plasmid, called TDNA, is transferred to the plant nuclear DNA where it is stably maintained and transcribed into mRNA. The TDNA codes for several functions which are expressed by the transformed plant cell. These functions include tumorigenesis, an effecting of hormone levels and bacterial metabolite (opine) synthesis.
The Ti plasmids are classified into two groups according to the number of host plants they can infect. A bacterial strain containing a wide host-range Ti plasmid can infect many dicotyledonous angiosperms as well as several gymnosperms. DeCleene, M. et al, Botan. Rev. 42, 389 (1976). However, each strain of A. tumefaciens has an overlapping but not necessarily identical host-range with other strains. This trait is primarily determined by the type of Ti plasmid present in the strain. Not all plants, including many dicotyledonous angiosperms and many gymnosperms, can be infected by A. tumefaciens.
Ti plasmids can be manipulated as gene vectors and have been used for the stable introduction and expression of foreign genes in plant cells and regenerated plants. See, for example, Fraley, R. T. et al, Proc. Nat. Acad. Sci. USA 84, 4803 (1983); Herrera-Estrella, L. et al. EMBO J 2, 987 (1983); Helmer, G. et al, Bio/Technology 2, 520 (1984); and Murai, N. et al, Science 222, 476 (1983). The key is to find a Ti plasmid which can infect the desired plant cell. This Ti plasmid can then be manipulated using standard techniques to enable the genetic engineering of the desired plant. The key element of the Ti plasmid is the vir region, which appears to be species-specific. In addition, TDNA, which does not appear to be species-specific, must be present to enable transfer of the desired DNA into the plant genome. These functions can be contained on separate plasmids within the transforming strain of A. tumefaciens.
The application of genetic engineering to conifers is in the initial stages. Included among the conifers are members of the family Pinaceae, including members of the genus Pinus. Very little information is presently available concerning the transformation of Pinaceae, especially Pinus, species. DeCleene, M., et al, supra, describe the infection of several members of the family Pinaceae by the A. tumefaciens strain B6. However, B6 was not able to infect any members of the genus Pinus. Prior to the present invention, no strain of A. tumefaciens was known to infect members of the genus Pinus.
U.S. Pat. No. 4,459,355 describes a method for transforming plant cells. The method involves the use of any A. tumefaciens which can transfer its Ti plasmid to the plant cell which is to be transformed. The patent indicates that the plant cell could come from a dicotyledon such as tobacco and tomato, or from a gymnosperm such as loblolly pine, cedar and Douglas fir. However, no transformation of loblolly pine is shown. The only example is directed to the transformation of tomato with the A. tumefaciens strain 15955. This strain is not capable of infecting pine. Furthermore, prior to the present invention, no strain of A. tumefaciens was known to infect pine, i.e., genus Pinus, even though strains were known to infect cedar and Douglas fir.
The present invention provides a method for transforming pine. Strains of A. tumefaciens have been identified which are capable of transforming pine. Additional strains can be identified by using the procedures described herein. All of these strains are useful for genetically engineering pine.